...the appropriate ligand - estradiol for hER or progesterone for hPR - leads to nuclear localization, binding...controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well as reporters...with various concentrations of estradiol and progesterone and showed that reporter expression is dependent...cognate inducer. Since induction via estradiol and progesterone do not affect each other, these systems can ...

...capable of producing a visible signal, but it has a strong affinity for avidin and streptavidin, both of which...modifying the antibody sequence or that rely on strong protein-protein interactions with other peptides...of non-canonical amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides... Ashley Waldron...

...sex and gender analysis important? Doing research wrong costs lives and money. According to the US General...treated, and other hormonal, immunological, and environmental factors. How to include sex in experimental ...Science, Health & Medicine, Engineering, and Environment—a project aimed at harnessing the creative power...Evoloving Check Out Extracurricular Activities for a Strong Science Career Path Additional Resources Check...

...effective at editing cytosines preceded by guanine (Thuronyi et al., 2019). Following the development of adenine...created a dual-deaminase editor called SPACE (synchronous programmable adenine and cytosine editor) by ...high product purity and low off-target editing Thuronyi et al., 2019 evoAPOBEC1-BE4max   Improved editing... K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C., Koblan, L. W., Zeng, J., Bauer...891. https://doi.org/10.1038/s41587-020-0453-z Thuronyi, B. W., Koblan, L. W., Levy, J. M., Yeh, W.-H....

...and Memory Formation. Neuron 79, 6 P1086-1093. https://doi.org/10.1016/j.neuron.2013.08.032.  Morita S...enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they resemble, actually impede...unmethylated CpG to behave naturally in the nuclear environment. One good system to balance these needs is the...

...in green monkeys and cultured human neurons, with broader neuronal expression than AAV9. This new capsid...-brain barrier and delivering a transgene, with neuron bias observed in young macaques and vascular bias...Recombinant anti-FLAG tag antibodies By Ashley Waldron Of all the epitope tags we know and love, the FLAG...

.... Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or optogenetics... Use of channelrhodopsin for activation of CNS neurons. Current protocols in neuroscience / editorial ... Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics... et al. A simple head-mountable LED device for chronic stimulation of optogenetic molecules in freely ...

... relaxed phone call than when first confronted on a stage in front of a crowd. The moderator will then...logistics To make a really lively discussion, I strongly suggest asking the panelists not to use any slides... turnout). This gets people to sit close to the front so the interactions are better. Have a good microphone...

...species, such as the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. ...CRISPRi in many types of bacteria: lab-adapted, environmental isolated, and disease-associated. Mobile-CRISPRi...non-model bacteria including pathogenic strains and environmental isolates. For example, Peters and colleagues... remained constant, with fitness effects more pronounced at 12 doublings than at 6. Key takeaways of Mobile-CRISPRi...

... to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations...CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a compact...selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use AAV vectors expressing...this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f are now available as a...

...mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery...tagging endogenous proteins with an auxin-inducible degron (AID) tag to generate proteins which can be rapidly...Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15... Michelle Cronin...

...many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of ... matter of concentration Most FPs in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form ... fused to the POI. These structures are large (microns), bright, non-physiological, and difficult to ignore...CFP and YFP dimers can form in the absence of environmental changes that the biosensor is designed to detect...

...change their excitation/emission behavior upon environmental changes (e.g. pH, Ca2+ flux, etc). The most ...Oligomerization: The first generations of FPs were prone to oligomerize. This may affect the biological function... these FPs cannot be used in oxygen deprived environment. Recently, a new GFP isolated from the Unagi ...you are planning to express the FP in acidic environments (e.g. yeast cytosol, which is slightly acidic...

...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any guesses? That's right... ways...genome-containing viral capsids is typically quantified by electron microscopy of a viral vector solution. Since this... this technique is intensive and requires an electron microscope, it is not routinely performed on all...genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene AAV viral vector preparation...

...experimental systems. They demonstrated LOV-Turbo in neurons, in pulse-chase experiments, and more. Find LOV-Turbo...element (TRE) or the cFos promoter. For example, neurons treated with a phorbol ester activator gave fluorescent...Images of iPAK4 fibers in cultured rat hippocampal neurons. HaloTag dyes are switched from yellow to red at... Teaching an old antibody new tricks By Ashley Waldron While IHC protocols typically call for thin sections...

...Boyden (Addgene viral prep # 84445-AAVrg).   Electron Microscopy The ratio of empty to full (i.e., genome-containing...representative vector preparations was quantified with electron microscopy after negative staining. Empty vector...than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation shows that...psPAX2 and pMD2.G packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing...

...refresher if you ever lose track of which acronym is which. Acronym Full name Notes   Base transition ...close at hand? Once again, this post has a lot of acronyms, so we've included a glossary at the end of this... for saturation mutagenesis, and they provide a strong proof of concept for further development. ...

...contains iterons that serve as recognition sites for replication initiation proteins. These iterons vary ...plasmid with multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad...Development of constitutive and IPTG-inducible integron promoter-based expression systems for Escherichia...

...pluripotent stem cells (Chavez et al., 2015) into neuronal cells by activating genes that cause differentiation...CRISPR-based activation can be made inducible upon environmental triggers. This is done by creating a split dCas9...CRISPR activation are also reversible: once the environmental trigger leaves, gene expression is no longer...Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez J, Yusa K, Garnett MJ (2019...

...either DTT or β-mercaptoethanol (BME). DTT is a stronger reducing agent than BME and has a less… distinctive...life when stored properly. Because BME is not as strong as DTT, you’ll have to use different concentrations...quantifying your blot. CCD cameras have a much higher upfront cost, but require no consumables, produce digital...like Coomassie Blue or Ponceau S. It is far less prone to error than normalizing to so-called housekeeping...happy blotting! Many thanks to Addgenies Ashley Waldron, Meghan Rego, and Amrita Rhodes for their help ...